Embryonal carcinoma cells, the stem cells of teratocarcinomas, are considered to be the tumorigenic equivalent of early embryonic cells. F9 is a murine embryonal carcinoma cell line which displays a low spontaneous rate of differentiation in vitro. However, trans-retinoic acid (RA) induces these cells to differentiate and to lose their tumorigenic phenotype. We demonstrated that a single F9 cell treated with RA has the potential to differentiate into at least three distinctive cell types. We refer to these cell types as EC', DifA, and DifB. Furthermore, we have isolated two F9 variants whose differentiation is restricted to particular stages of this pathway. The 5C variant cells treated with Ra differentiate exclusively into EC' cells. Under similar conditions the 5CRev variant cells differentiate into EC' and DifA cells, but not DifB cells. RA does not suppress the tumorigenic phenotype of these variant cells. We identified two other agents which induce differentiation of F9 cells--5-bromodeoxyuridine and N'-N'-dimethylacetamide. In both cases, differentiation is closely coupled with a loss of the tumorigenic phenotype, indicating that the regulation of these two events is closely coupled in some fashion. (M)